Tuberculosis (TB) is the leading infectious killer of youth and adults and the first most common infectious disease worldwide. One third of the world's population is currently infected and 20 million of those infected are active cases. TB will kill 30 million people this decade. More than 50 million people may already be infected with multidrug-resistant (MDR) strains of TB. TB is now becoming the leading cause of death among HIV positive people where it kills much more rapidly with a fatality of 80%.
Tuberculosis is caused by infection with M. tuberculosis, a bacillus bacterium. It is spread by aerosol droplets and causes irreversible lung destruction. If it escapes the lung it may cause systemic disease affecting many organs including bones, joints, liver, spleen, gastrointestinal tract and brain. 50% of people exposed to M. tuberculosis are infected with the bacteria and 15% of those infected develop disease. Poverty, malnutrition and overpopulation contribute dramatically to the perseverance and wild spread of tuberculosis.
Past means of controlling TB have involved the use of combinations of antibiotics. Recently, because of complications due to MDR strains, the number and combination of antibiotics administered must be individually tailored depending on the strain the patient is harboring. In extreme cases, surgical removal of the infected portion of the lung is required.
Traditionally, the diagnosis of TB has been made on the basis of clinical findings and chest radiographs and confirmed by sputum or tissue smears that show TB bacilli. These methods remain the “gold standard” for diagnosis, but development of DNA probes, polymerase chain reaction (PCR) assays, and liquid media now allow more sensitive and rapid diagnosis. Unfortunately, increased sensitivity of rapid techniques is not always associated with increased specificity.
The Amplified M. tuberculosis Direct Test (Gen-Probe) targets mycobacterial ribosomal RNA by transcription-mediated amplification. The test uses DNA probes that are highly specific for M. tuberculosis species. It is best used (and only approved for use) in patients in whom acid-fast bacilli smears are positive and cultures are in process. Since specificity is less than 100%, even in patients with positive smears, occasional false-positive results do occur, usually in patients with nontuberculous mycobacterial infections.
W. C. Yam et al, published “DIRECT DETECTION OF MYCOBACTERIUM TUBERCULOSIS IN CLINICAL SPECIMENS USING SINGLE-TUBE BIONTINYLATED NESTED POLYMERASE CHAIN REACTION-ENZYME LINKED IMMUNOASSAY (PCR-ELISA)” in Diagnostic Microbiology and Infection Disease, 48 (2004) p. 271-275, provided an improved assay for detecting M. tuberculosis. However, it takes around 2 hours for performing hot start to activate Taq polymerase and the amplification for the outer and inner PCR products.
The trend of diagnostic laboraory medicine is automation, and nucleic acid amplification assays are no exception to this rule. The known method (such as real-time PCR assay) using the LightCycler (LC) instrument provides a rapid, sensitive and specific means to identify M. tuberculosis. However, the use of LC as a routine diagnosis of M. tuberculosis remains limited at present. It is because the installment and maintenance cost may not be afforded by most of the center.
It still has a method, system and kit to provide a simple, low-cost, accurate, high throughput and saving-labor and operation time to detect DNA of M. tuberculosis in a sample with comparable sensitivity and specificity with commercial products.